The Detection Of Extended-spectrum β-lactamase In Pseudomonas Aeruginosa

Background:With the broad extensive application of the spectrum antibiotic, the bacterial drug resistance is a tough problem in current infection territory. Especially with the bacterial infection, drug resistant strain and drug resistance becoming more and more multiplicity in the past few years, the control and therapy of infections diseases are severely influenced and hindered, and the medical cost is greatly increased .So the distribution transition and drug sensitivity of bacterium are important in the control of infectious diseases and the reasonable using of antibiotics.Pseudomonas aeruginosa whether whom causing the infection of hospital infection mainly, mechanism of drug-resistance to be complicated, it has natural drug-resistance and also easily obtaining acquired drug-resistance after using antibiotic, it causes great difficulty to clinical therapy.Along with the generally utilizing of tert-cephalosporin on clinic, the drug resistance gene of β-lactamase in bacterium happen saltation, produce new β-lactamase, it can also hydrolysis superspectrum cephalosporin plainly besides can hydrolysis already existing β-lactam antibiotics, so call it extended -spectrum β-lactamases(ESBLs).The plasmid taking along ESBLs can make its sudden changed gene transmitted to with planting or different kind of bacterial strain, make more bacteria produce drug-resistance, thus cause endangering to treating clinically greatly. A lot of produce ESBLs bacterial strain display sensitiveness to susceptibility test outside body, but able to bearing medicines clinically, thus delay treating, and because ESBLs can be being carried by the plasmid, it is very apt to transmit andspread among bacterium, cause the extensively spreading of drug-resistance. So, it is essential to examine out ESBLs accurately as soon as possible.NCCLS1999 annual edition of ESBLs phenotypes issued screens and confirms experiment is the most extensive ESBLs detection method at present, phenotype characteristic that the principle can be suppressed by pMactamase inhibitor on the basis of ESBLs; Produce ESBLs to be clinical to separate often also with other complicated mechanisms of drug-resistance, so has limited this method in the clinical application while measuring, Other detection methods are like E-test law, three-dimension experimental law, Vitek machine determine law wait too because experiment method miscellaneous and equipment reason such as being expensive limit in clinical to measure popularization of, so set up one peculiar sensitive suitable detection system for most Gram-negative bacteria clinical is a urgent task.Part IThe investigation and analysis of pathogens in ICU ward hospital of General hospital of Military Area Command ofGuangzhou in 1999 - 2003Objectives:Through analyzing the kind of infect bacterium and the result of drug sensitivity test, comprehend the distribution of the clinical bacterial infection and the transmutation of. drug-resistance in General hospital of Military Area Command of Guangzhou in 1999 - 2003. Methods:1 Collect sample that such position as various kinds of drainage liquid and surgical wound secretion etc. are adopted of patient in ICU.2 Adopt VITEK microorganism's automatic detector to measure and appraise the experiment bacterial strain goes on with k-b law that NCCLS recommends. Judge standard and result explain consult standard, NCCLS of U.S.A., (1999 edition).3 All initial data are analyzed by WHONET4 software that the monitoring net of WHO bacterial drug resistance offering.Results:1.Bacterium distributed take Gram-negative as the core, Pseudomonas aeruginosa is a main bacterium causing the hospital infection , Gram positive coccus is mainly Staphylococcus aurous, and methicillin resistant staphylococcus aurous (MRSA )accounts for 76.3% .2 The multidrug resistant of MRSA compared with report in the past increases to some extent, antibiotic medicine sensitive test of Pseudomonas aeruginosa appears increasing trend too , Imipenem is still sensitive to it. Conclusions:1 The general hospital of Military Area Command of Guangzhou infects bacteria still with Gram-negative main fact, take Pseudomonas aeruginosa as the core among them, production of MRSA increase to some extent.2 The drug resistance of MRSA increases to some extent; Imipenem is still a first-selected medicine to treat Pseudomonas aeruginosa.Part II Detection of the extended-spectrum P-lactamasein Pseudomonas aeruginosa Objectives:Through the detection of pseudomonas aeruginosa clinical separation which suspect of produces the extended-spectrum P-lactamase, to understand the distribution of ESBLs genotype in the general hospital of Military Area Command of Guangzhou; to appraisal the application of dual PCR in ESBLs detection. Methods:1. The medicine sensitive experiment was carried on by double slip paper coordination experiment and confirms experiment2. Template preparation and DNA purification;3. The TEM, SHV genes were amplificated by dual PCR, the genotypes were analyzed by electrophoresis. Results:Of the 30 Pseudomonas aeruginosa clinical separation which produce the extended-spectrum P-lactamase, 21 of which were proved to have TEM gene and 3 have SHV gene. Conclusions:Dual PCR law contributes to improving examining the appearing rate of ESBLs gene, it is completion and supplement to that NCCLS phenotype screened and confirmed the experiment .The Pseudomonas aeruginosa clinical separation which produce the extended-spectrum P-lactamase in the general hospital of Military Area Command of Guangzhou was mainly interposed by SHV and TEM gene.